In recent years, as a result of environmental problems such as global warming and atmospheric pollution, as well as concerns related to energy supplies for transportation, including the dramatic increase in the cost of crude oil and the expectation of a depletion in crude oil sources in the near future (peak oil), the development of alternative energy sources to oil has become an extremely important issue. Plant biomass or lignocellulose is the most plentiful renewable energy source on earth, and holds great promise as an alternative energy source to oil. The main component of plant biomass dry weight is lignocellulose, which is composed of polysaccharides such as cellulose and hemicellulose, and lignin. For example, polysaccharides can be hydrolyzed by a glycoside hydrolase such as a cellulase or hemicellulase to form monosaccharides such as glucose and xylose, which can then be used as biofuels or the raw materials for chemical products.
Lignocellulose is recalcitrant due to its highly complex structure, and is difficult to degrade or hydrolyze with a single glycoside hydrolase. The complete degradation of lignocellulose generally requires three types of enzymes, namely an endoglucanase (cellulase or endo-1,4-(3-D-glucanase, EC 3.2.1.4), an exo-type cellobiohydrolase (1,4-β-cellobiosidase or cellobiohydrolase, EC 3.2.1.91, EC 3.2.1.176), and a β-glucosidase (EC 3.2.1.21), and it is thought that the addition of a further plurality of enzymes including the hemicellulase xylanase (endo-1,4-β-xylanase, EC 3.2.1.8) and other plant cell wall-degrading enzymes such as β-xylosidase (EC 3.2.1.37) is also necessary.
In conventional bioethanol production using lignocellulose as a starting resource, hydrolysis processes using high solid loading (30 to 60% solid loading) have been tested with the aim of achieving a more energy-efficient conversion to ethanol. However, in this type of lignocellulose enzymatic hydrolysis using high solid loading, the viscosity of the hydrolyzed biomass solution is high, and the hydrolysis reaction of the lignocellulose tends to proceed poorly. Accordingly, by using a thermostable enzyme and performing the enzymatic hydrolysis process at a high temperature, for example 65° C. or higher, the rate of the hydrolysis reaction can be increased, and the viscosity of the hydrolyzed biomass solution can be reduced, which is expected to enable a shortening of the hydrolysis reaction time and a reduction in the amount of enzyme required. Another advantage is that by performing the reaction at high temperature, proliferation of unwanted bacteria during the enzyme reaction can be prevented. As a result, for all of the various glycoside hydrolases, the development of enzymes having superior thermal stability is very desirable.
Cellulases that function in high-temperature environments have conventionally been isolated from thermophilic filamentous fungi and thermophilic bacteria and the like, but the majority of these cellulases are enzymes having endoglucanase activity, xylanase activity, xylosidase activity or glucosidase activity, and very few cellobiohydrolases, which play an important role in lignocellulose hydrolysis processes, have been isolated. However, in terms of cellobiohydrolases which initiate hydrolysis from the non-reducing ends of cellulose, a cellobiohydrolase of the GH6 family having an optimum temperature exceeding 75° C. has been reported (for example, see Patent Document 1).
Among cellobiohydrolases, there are some enzymes which are composed of not only the catalytic domain that hydrolyzes cellulose, but also have a module that has the function of binding cellulose (hereafter sometimes referred to as a “carbohydrate-binding module” or CBM). Although the CBM itself exhibits no degradation activity, the CBM by itself has the ability to bind to cellulose. Known functions of CBMs include increasing the concentration of the catalytic domain in the vicinity of the substrate by adsorbing to the insoluble substrate, thereby increasing the cellulose degradation rate, and severing hydrogen bonding between cellulose chains through CBM binding, thereby destroying crystal structures (Non-Patent Documents 1 and 2). Further, if a CBM is removed from a cellobiohydrolase which degrades crystalline cellulose, then although the reactivity relative to soluble substrates does not change, the degradation activity and affinity relative to crystalline cellulose decrease dramatically, and therefore it is thought that the CBM is a domain that is required for the enzyme to act upon crystalline cellulose (Non-Patent Document 3).